WO2006002547A1 - Adaptateur d'acide nucleique dependant de la cible - Google Patents

Adaptateur d'acide nucleique dependant de la cible Download PDF

Info

Publication number
WO2006002547A1
WO2006002547A1 PCT/CA2005/001051 CA2005001051W WO2006002547A1 WO 2006002547 A1 WO2006002547 A1 WO 2006002547A1 CA 2005001051 W CA2005001051 W CA 2005001051W WO 2006002547 A1 WO2006002547 A1 WO 2006002547A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
nucleic acid
stem
target
biosensor
Prior art date
Application number
PCT/CA2005/001051
Other languages
English (en)
Inventor
Jean-Pierre Perreault
Lucien Junior Bergeron
Original Assignee
UNIVERSITé DE SHERBROOKE
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by UNIVERSITé DE SHERBROOKE filed Critical UNIVERSITé DE SHERBROOKE
Priority to EP20050761763 priority Critical patent/EP1814896A4/fr
Priority to US11/631,689 priority patent/US9012140B2/en
Priority to CA2632216A priority patent/CA2632216C/fr
Publication of WO2006002547A1 publication Critical patent/WO2006002547A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/123—Hepatitis delta
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3519—Fusion with another nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity

Definitions

  • the present invention relates to the field of target-dependent switch adapters for nucleic acid sequences, and more particularly to adapters for nucleic acid sequences such as ribozymes.
  • RNA has a series of distinct capabilities and biological activities previously unsuspected.
  • the most important of these novel RNA-level discoveries has been the finding that RNA can be an enzyme as well as an information carrier.
  • RNA molecules have one or more enzymatic activities, such as an endoribonuclease activity which acts to cleave other RNA molecules. Such activity is termed intermolecular cleaving activity.
  • enzymatic RNA molecules are derived from an RNA molecule which has an activity which results in its own cleavage and splicing. Such self-cleavage is an example of an intramolecular cleaving activity.
  • RNA-based pathogenic agents include the lethal Acquired Immune Deficiency Syndrome (AIDS) and delta hepatitis (also called Hepatitis D), a particularly virulent form of fulminant hepatitis caused by a viroid-like RNA agent.
  • AIDS lethal Acquired Immune Deficiency Syndrome
  • delta hepatitis also called Hepatitis D
  • RNA-borne diseases are spread at the RNA level, manifest themselves in cells of patients, and are by now present within the bloodstream of millions of individuals.
  • ribozymes RNA enzymes
  • Ribozymes are an interesting alternative to RNA interference approach that seems to trigger immunological responses.
  • Many efforts were directed at increasing the substrate specificity of ribozyme cleavage, which can be considered as a limit to their utilization.
  • allosteric ribozymes for which the catalytic activity is regulated by an independent effector have been developed.
  • Delta ribozymes derived from the genome of hepatitis delta virus (HDV), are metalloenzymes. Like other catalytically active ribozymes, namely hammerhead and hairpin ribozymes, the delta ribozymes cleave a phosphodiester bond of their RNA substrates and give rise to reaction products containing a 5'-hydroxyl and a 2',3'-cyclic phosphate termini. Two forms of delta ribozymes, namely genomic and antigenomic, were derived and referred to by the polarity of HDV genome from which the ribozyme was generated.
  • delta ribozymes have a potential application in gene therapy in which an engineered ribozyme is directed to inhibit gene expression by targeting either a specific mRNA or viral RNA molecule.
  • a very low concentration ( ⁇ 0.1 mM) of Ca 2+ and Mg 2+ is required for delta ribozyme cleavage.
  • the ⁇ ribozyme folds into a compact secondary structure that includes pseudoknots (for reviews see Bergeron et al., Current Med. Chem. 10, 2589-2597, 2003).
  • This structure is composed of one stem (the P1 stem), one pseudoknot (the P2 stem is a pseudoknot in the cis- acting version), two stem-loops (P3-L3 and P4-L4) and three single-stranded junctions (J 1/2, J 1/4 and J4/2). Both the J 1/4 junction and the L3 loop are single-stranded in the initial stages of folding, but are subsequently involved in the formation of a second pseudoknot that consists of two Watson-Crick base pairs (the P1.1 stem).
  • the P1 and P3 stems, along with the J4/2 junction form the catalytic center, while the P2 and P4 stems are located on either side of the catalytic centre and stabilize the overall structure.
  • the binding domain of ⁇ Rz (the P1 stem) is composed of one G-U wobble base pair followed by six Watson-Crick base pairs.
  • the nucleotides from position -1 to -4 of the substrate that is those adjacent to the scissile phosphate, were shown to contribute to the ability of a substrate to be cleaved efficiently.
  • the substrate specificity of ⁇ Rz cleavage is based on a total of 11 nucleotides, which might be a limiting factor when trying to specifically target an RNA species in a cell. Because the P1 stem is located - A - within its catalytic center, all attempts to modify the length of this stem result in the loss of catalytic ability.
  • ribozymes derived from a specific domain present in the hepatitis delta virus (HDV) RNA for specifically cleaving targeted RNA sequences and uses thereof for the treatment of disease conditions which involve RNA expression, such as AIDS.
  • HDV hepatitis delta virus
  • These ribozymes consist in at least 18 consecutive nucleotides from the conserved region of the hepatitis delta virus between residues 611 and 771 on the genomic strand and between residues 845 and 980 on the complementary anti-genomic strand. These ribozymes are proposed to fold into an axe-head model secondary structure.
  • these ribozymes require substrate base paired by 12-15 nucleotides. More specifically, a substrate bound to the ribozyme through the formation of two helices. A helix is located upstream to the cleavage site (i.e. in 5' position) while the second helix is located downstream to the cleavage site (i.e. in 3' position).
  • the specificity of recognition of these ribozymes is limited to 6 or 7 base pairing nucleotides with the substrate and a preference of the first nucleotide located 5' to the cleavage site. Neither tertiary interaction(s) between the base paired nucleotides and another region of the ribozyme, nor single-stranded nucleotides are involved to define the specificity of recognition of these ribozymes. Because the recognition features were included in a very small domain (i.e. 6 or 7 base paired nucleotides) in order to exhibit the desired activity, these ribozymes have a limited specificity, and thus, not practical for further clinical applications.
  • One aim of the present invention is to provide a new ribonucleic acid, target-dependent adapter to increase efficiency and specificity (prevents cleavage of an inappropriate target) of cleavage of a ribonucleic acid for its target.
  • the adapter of the present invention can be used as a switch to turn on or off a ribonucleic acid enzyme by controlling availability of the target of this enzyme. Whenever the target is not available, the adapter turns off the enzyme by adopting an inactive conformation and when the substrate or the target is detected by the adapter, the enzyme is turned on in an active conformation.
  • nucleic acid when linked to the adapter would be hidden (in an inactive conformation) or prevented to react with anything else in absence of a target and would be made available to react with its target upon detection of said target by the adapter.
  • the switch of the present invention can be modified to be adapted to any nucleic acid sequences that target a substrate, making the switch a new versatile and powerful tool, allowing to increase the specificity of the nucleic acid for its substrate or target, also allowing to increase the cleavage efficacy of the ribozyme for its substrate or target, and to abolish the non-specific pairing therefore reducing false positive reactions.
  • a target-dependent nucleic acid adapter adapted to be matched to a substrate comprising a target sequence, said adapter having a nucleic acid sequence comprising linked together:
  • a blocker stem sequence complementary to a portion of said nucleic acid sequence i) a blocker stem sequence complementary to a portion of said nucleic acid sequence; and N) a biosensor sequence having a sequence complementary to said target sequence, said biosensor improving the specificity of the nucleic acid sequence for said target sequence,
  • said blocker stem sequence forms an intramolecular stem with said nucleic acid sequence linked thereto, preventing exposition of the nucleic acid sequence, thus locking said nucleic acid sequence of the adapter in an inactive conformation, and, in presence of said target sequence of said substrate, said biosensor sequence forming conventional Watson-Crick base pairs with said target sequence and said blocker stem sequence dissociating from the intramolecular stem, thus exposing said nucleic acid sequence of said adapter in an active conformation.
  • the target dependent nucleic acid adapter may also comprises sequences forming a stabilizing stem, whereby the 3'-end of the adapter so linked to said nucleic acid sequence is paired up, thus preventing or reducing degradation of said nucleic acid sequence.
  • the stabilizing stem may have for example two complementary strands, a first strand of which is linked to the 5'- end of the biosensor, and a second strand of which that is complementary to the first strand and that is adapted to be linked at its 5'-end to the 3'-end of the nucleic acid sequence, thus preventing exposure of a single stranded 3'-end sequence susceptible to degradation by cellular nuclease.
  • the first strand of the stabilizing stem has a sequence as set forth from residue 4 to 11 of SEQ ID NO:1 and the second strand of the stabilizing stem has a sequence as set forth from residue 96 to 103 of SEQ ID NO:1.
  • the blocker stem sequence has a sequence specific for a ribozyme, such as ribozyme delta.
  • the biosensor has a sequence as set forth from residue 15 to 29 of SEQ ID NO:1.
  • the blocker stem sequence has in one embodiment a sequence as set forth from residue 30 to 33 of SEQ
  • the blocker stem sequence is linked downstream of the biosensor.
  • a target-dependent nucleic acid adapter having a nucleic acid sequence comprising:
  • biosensor sequence having a sequence complementary to said target sequence, said biosensor improving the specificity of the nucleic acid sequence for said target sequence
  • said blocker stem sequence forms an intramolecular stem with said nucleic acid sequence linked thereto, preventing exposition of the nucleic acid sequence, thus locking said nucleic acid sequence of the adapter in an inactive conformation, and, in presence of said target sequence of said substrate, said biosensor sequence forming conventional Watson-Crick base pairs with said target sequence and said blocker stem sequence dissociating from the intramolecular stem, thus exposing said nucleic acid sequence of said adapter in an active conformation.
  • a method for turning on or off an enzymatic activity of a nucleic acid molecule having an enzymatic activity comprising the steps of attaching to said nucleic acid molecule a nucleic acid target dependent adapter having a nucleic acid sequence comprising:
  • biosensor sequence having a sequence complementary to said target sequence, said biosensor improving the specificity of the nucleic acid sequence for said target sequence
  • said blocker stem sequence forms an intramolecular stem with said nucleic acid sequence linked thereto, preventing exposition of the nucleic acid sequence, thus locking said nucleic acid sequence of the adapter in an inactive conformation, turning off the enzymatic activity and, in presence of said target sequence of said substrate, said biosensor sequence forming conventional Watson-Crick base pairs with said target sequence and said blocker stem sequence dissociating from the intramolecular stem, thus exposing said nucleic acid sequence of said adapter in an active conformation, turning on the enzymatic activity.
  • a target-specific activatable/deactivatable ribonuclease adapted to be matched to a substrate comprising a target sequence, said ribonuclease having a nucleic acid sequence comprising linked together:
  • biosensor sequence having a sequence complementary to said target sequence, said biosensor improving the specificity of the ribonucleic acid sequence for said target sequence, said biosensor being linked to the blocker sequence,
  • said blocker sequence in absence of the target sequence of said substrate, said blocker sequence forms an intramolecular stem with the ribonuclease sequence linked thereto, thus locking said ribonuclease in an inactive conformation, and, in presence of the target sequence of said substrate, said biosensor sequence forming conventional Watson-Crick base pairs with said target sequence and said blocker stem sequence dissociating from the intramolecular stem, thus exposing said ribonuclease in an active conformation.
  • the target-specific activatable/deactivatable ribonuclease has a sequence as set forth in SEQ ID NO:1.
  • RNA with enzymatic or effector activity is intended to mean any RNA that has an active and inactive conformation or any RNA that has an enzymatic activity or that has an effect on either the transcription of said target RNA or a downstream event following transcription of said RNA.
  • substrate can be substituted by "target” or “target substrate” or the expression “substrate or target” throughout the application. It is to be recognized and understood that the substrate contains a target sequence.
  • Biosensor can be abbreviated as “BS” or “BSO”
  • SOFA module can be substituted by the term "SOFA adapter" throughout the application.
  • Target dependant nucleic acid adapter can be substituted by the term “nucleic acid target dependent adapter” throughout the application.
  • a method for turning on/off an enzymatic activity of a nucleic acid molecule having an enzymatic activity can be adapted to any type of nucleic acid enzymes catalyzing the modification of nucleic acid substrates (i.e. modifying enzymes such as kinases, ligases, methylases, ribonucleases, aminoacyl-tRNA synthetases, etc).
  • modifying enzymes such as kinases, ligases, methylases, ribonucleases, aminoacyl-tRNA synthetases, etc.
  • Fig. 1 is a schematic representation of both the off and on conformations of the SOFA-ribozyme of the present invention
  • Figs. 2A and 2B illustrate the specific structure and sequence of the SOFA-ribozyme (SEQ ID NO:1 ), in the off (Fig. 2A) and on (Fig. 2B) conformations, in accordance with one embodiment of the present invention, where the arrow in the on conformation indicates the cleavage site on the target
  • Fig. 3 illustrates an autoradiogram of a denaturing 6% PAGE gel for the analysis of the cleavage reaction of the HBV-derived target by the original- (Wild Type) (WT), SOFA- ⁇ Rz-303 and SOFA- ⁇ Rz-513 ribozymes;
  • Fig. 4 illustrates a graphical representation of time courses for the cleavage reactions of SOFA + (squares), SOFA " (circles) and the original (inversed triangles) ribozyme versions of ⁇ Rz-303 (filled) and ⁇ Rz-513 (empty);
  • Fig. 5 illustrates an autoradiogram of a denaturing 6% PAGE gel showing the cleavage assays of the HBV-derived substrate by SOFA- ⁇ Rz-303 bearing a biosensor stem of various lengths (BS-X, where X indicates the length of the stem) to characterize the SOFA- ⁇ Rz-303;
  • Fig. 6 illustrates an autoradiogram of a 20% PAGE gel showing the cleavage assays of the HBV-derived substrate of 44 nucleotides by a SOFA- ⁇ Rz-303 bearing a biosensor stem of various lengths (BS-X, where X indicates the length of the stem);
  • Figs. 7A and 7B illustrate an analysis of the mechanism of action of the SOFA-ribozyme, showing the proposed sequential interactions between the ribozyme and the substrate (Fig.
  • Figs. 8A to 8F illustrate an analysis of the substrate specificity of various SOFA-ribozymes, where Figs. 8A to 8C show a schematic representation of the various substrates, while Figs. 8D to 8F illustrate the autoradiograms of denaturing 6% or 20% PAGE gels performed for these cleavage assays;
  • Fig. 9 illustrates the sequences of all the targeting sites used in Figs. 1 to 8 except in Figs. 8A and 8D;
  • Fig. 1OA illustrates the stem formed between 8 pairs of substrate (a to h, left) and ribozyme biosensor (A to H, right) sequences
  • Fig. 1OB and 1OC illustrate the autoradiogram of a typical 10% denaturing PAGE gel of a time course experiment performed under single turnover conditions for the pair Dd, and the graphical representation of the time course of ribozyme D cleaving each of the substrates (a to h);
  • Fig. 1OD illustrates the histogram of the k O bs values for each of the 64 possible pairs
  • Fig. 11A illustrates twenty-three biosensor sequence variants examined for their ability to cleave the short 44 nt HBV-derived substrate.
  • the mutations are boxed in grey, and the k O bs values (in min '1 ) are indicated on the right.
  • the stars indicate the SOFA- ⁇ Rz-303 mutants for which the k cat , and KM values were determined for the cleavage of a long version of the HBV-derived substrate (1 190 nt);
  • Fig. 1 1 B illustrates the average values of k O b S from at least two independent sets of experiments for each cluster of mutated ribozymes.
  • Fig. 12A illustrates four blocker stems tested
  • Fig. 12B illustrates an autoradiogram of a 6% denaturing PAGE of the cleavage assays performed with the SOFA- ⁇ Rz-303 variants possessing mutated blocker sequences (i.e. BL-X, where X indicates the size of the blocker stem).
  • the reactions were performed under single turnover conditions using the 1190-HBV substrate. The sizes of the bands are indicated on the right of the gel.
  • the control (-) was performed in the absence of ribozyme;
  • Fig. 12C illustrates a kinetic analysis performed for each of the mutants:
  • BL-O squares
  • BL-2 circles
  • BL-4 inverse triangles
  • BL-5 diamonds.
  • Fig. 13A illustrates the design of the substrates used to analyze the importance of the spacer sequence.
  • the substrate P1 strand of SOFA- ⁇ Rz-303 was repeated seven times (P1 N , 1 -7) within seven substrates possessing spacers of different sizes (0 to 6 nt);
  • Fig. 13B illustrates an autoradiogram of a 10% denaturing PAGE of the cleavage assays performed with each of the seven substrates.
  • Lanes 0 to 6 correspond to the different sizes of the spacer sequences (i.e. from 0 to 6 nt).
  • the migrations of the substrates (S) and their sizes, as well as those of the cleavage products, are indicated adjacent to the gel.
  • XC and BPB indicate xylene cyanol and bromophenol blue;
  • Fig. 13C illustrates the relative percentage of cleavage as a function of spacer length.
  • the bracket indicates the optimal length (1 to 5 nt), and dashed lines separate the observed transitions;
  • Fig. 13D illustrates the histogram of the relative percentage of cleavage of the substrates possessing spacers of various lengths (5, 19, 33 and 47 nt).
  • the inset shows the autoradiogram of the corresponding 10% denaturing PAGE gel
  • Fig. 14A illustrates an autoradiogram of a 6% denaturing PAGE of cleavage assays of various SOFA- ⁇ Rz-303 variants synthesized to evaluate the importance of the stabilizer sequence, where lane 1 is the incubation of the long HBV-derived substrate (1190 nt) alone, while lane 2 is that in the presence of the original ⁇ Rz-303, lanes 3 and 4 are the cleavage assays performed with
  • Fig. 14B illustrates the result obtained with mutated stabilizer.
  • the upper panel illustrates the sequence of the stabilizer (SOFA- ⁇ Rz-303-ST1 to -ST4), while the lower panel illustrates the autoradiogram of the 6% PAGE of the corresponding cleavage assays.
  • the control (-) was performed in the absence of ribozyme;
  • Fig. 15A illustrates a schematic representation of the SOFA-ribozyme in both the off and on conformations, where the on conformation is obtained after the addition of the substrate.
  • the bold lines indicate the oligodeoxynucleotides.
  • Fig. 15B illustrates an autoradiogram of an 8% denaturing PAGE of the probing assay.
  • the symbols (-) and (+) indicate the presence or absence, respectively, of the substrate for the probing performed using each oligodeoxynucleotide (L3 1 , P4 1 , BS', BL' and ST').
  • the positions of the expected cleavage products, XC and BPB are indicated adjacent to the gel.
  • Fig. 16A illustrates the expression vector of the HBV-derived gene C used in the in vivo cleavage assays of the HBV-derived substrate by SOFA- ⁇ Rz-303;
  • Fig. 16B illustrates the expression vector for various ribozyme versions in accordance with various embodiments of the present invention, used in the in vivo cleavage assays of the HBV-derived substrate by SOFA- ⁇ Rz-303;
  • Fig. 16C illustrates autoradiograms of a Northern blot hybridization performed after a denaturing 1.3% agarose gel where ⁇ -actin and HBV mRNAs were detected using 32 P-labelled RNA probes;
  • Figs. 17A 1 17B and 17C show sequences and secondary structures of the SOFA+- ⁇ Rz-Down (SOFA+- ⁇ Rz-DN) and SOFA+- ⁇ Rz-Double (SOFA+- ⁇ Rz- DB), demonstrating the versatility of the SOFA- ⁇ Rz-303.
  • Fig. 17D illustrates autoradiograms of denaturing 6% PAGE gels performed for these cleavage assays, including a control (-) performed in the absence of ribozyme;
  • Fig. 18A illustrates the sequence and secondary structure of the SOFA + - ⁇ Rz without the stabilizer (SOFA + - ⁇ Rz-NS, NS for no stabilizer);
  • Fig. 18B illustrates autoradiograms of 6 % denaturing PAGE gel of the cleavage assays
  • Fig. 19A to 19F illustrate the sequence and secondary structure of various SOFA-ribozymes and SOFA-DNazyme in accordance with one embodiment of the invention, showing both off and on conformations, wherein the small arrows of the on conformations indicate the cleavage or ligation sites;
  • Fig. 20 illustrates an autoradiogram of 6 % denaturing PAGE gel of the cleavage assays obtained for the SOFA-DNazyme
  • Figs. 21A to 21C illustrate the sequence of a SiRNA (Fig. 21A), the sequence and secondary structure of a SOFA-siRNA version in accordance with a further embodiment of the invention, showing both off (Fig. 21B) and on
  • RECTIFIED SHEET (RUI 4 E 91.1) module that acts as a biosensor (Fig. 1).
  • the ribozyme In absence of its target, the ribozyme is inactive (off), while in the presence of the desired target the biosensor recognizes it and activates (turns on) the ribozyme's cleavage activity.
  • SOFA Specific On/ofF Adapter
  • the SOFA (Box 8 on Figs. 2A and 2B) includes three domains, also called sequences or segments:
  • BS Sequence from ribonucleotide 15 to 29 of SEQ ID NO:1 on Figs. 2A and 2B; Box 12
  • the blocker 10 forms an intramolecular stem with the P1 strand (Sequence from ribonucleotide 55 to 61 of Figs. 2A and 2B; Box 18), thereby generating an inactive conformation.
  • the biosensor anneals with the substrate, thereby releasing the P1 strand so that it can subsequently hybridize with the substrate, initiating formation of the active conformation.
  • the target has two roles acting simultaneously, one as an activator and one as a substrate.
  • the biosensor acts as a riboswitch regulating the catalytic activity.
  • the stabilizer localizes the 3'-end of the SOFA module in a double-stranded region that stabilizes the ribozyme from the cellular nucleases (Levesque, D., et al., RNA 8, 464-477, 2002).
  • a switch also referred to herein as a Specific On/Off Adapter or SOFA to improve specificity of a nucleic acid sequence such as DNA or RNA for its target and/or control the activity of said nucleic acid sequence.
  • This construct can be made specific to particular ribozymes or RNA with enzymatic or effector activity, to activate or inactivate ribozymes or RNA simply by changing and matching the sequence of the biosensor with the complement of that of the target sequence, so that pairing up between the two can occur, when in presence of each other.
  • the biosensor must bind its complementary sequence on the substrate in order to unlock the SOFA module, thereby permitting the folding of the catalytic core into the on conformation.
  • Both the blocker and the biosensor have been shown to increase the substrate specificity of the ribozyme's cleavage by several orders of magnitude as compared to the wild-type ⁇ Rz. This is due mainly to the addition of the biosensor domain that increases the binding strength of the ⁇ Rz to its target, but is also due to the fact that the blocker domain interacts with the P1 region and decreases its binding capacity.
  • the stabilizer which has no effect on the cleavage activity, stabilizes the RNA molecule in vivo against ribonucleases. The purpose of the stabilizer sequence is to pair up the 3' end of the sequence to prevent degradation.
  • HBV pregenome insert (from pCHT-9/3091 , Nassal, M. J. Virol. 66, 4107-4116, 1992) was subcloned downstream of the T7 RNA promoter in the vector pBlueScript SKTM (Stratagene) using the Sa/I and Sac ⁇ restriction sites, and the resulting plasmid named pHBVT7 (Bergeron, LJ. , & Perreault, J. P. Nucleic Acids Res. 30, 4682-4691 , 2002).
  • An HBV 1190 nt fragment was excised from pCHT9/3091 using Sacl and EcoR ⁇ , and then subcloned into pBlueScript SKTM, generating pHBV-1190.
  • HBV 44 nt substrate was produced using a PCR-based strategy with T7 sense primer: 5'-TTAATACGAC TCACTATAGG G-3' (SEQ ID NO:3) and antisense primer: ⁇ '-CTTCCAAAAG TGAGACAAGA AATGTGAAAC CACAAGAGTT GCCCTATAGT
  • the plasmid pHCVA was obtained by cloning the 1348 nt HCV 5' sequence from pHCV-1b (Alaoui-lsmaili, et al., Antiviral Res. 46, 181-193, 2000) into Hind ⁇ and BamHl pre-digested pcDNA3 vector.
  • the original ⁇ ribozymes were constructed as described previously (Bergeron, L.J., & Perreault, J. P. Nucleic Acids Res. 30, 4682-4691, 2002). SOFA +/' ribozymes were constructed using a PCR-based strategy including two complementary and overlapping oligonucleotides.
  • the antisense primers were: 5'-AAAGTGAGACAAGAA-(A)o-6nr
  • AAAAAACCCTATAGTGAGT CGTATTAA-3' AAAAAACCCTATAGTGAGT CGTATTAA-3' (SEQ ID NO:15), where the T7 promoter sequence is underlined.
  • the antisense primers were: 5'- AAAGTGAGACAAGAA(AAAAC)SP-(ACCAACA)X(AAACCAC)Y(ACCAACA)Z-
  • AAAAAACCCTATAGTGAGTCGTATTAA-3' (SEQ ID NO: 16) (where SP is for spacer, the number of X and Z units varied as desired; the unit Y gives the cleavable P1 sequence; and, the T7 promoter sequence is underlined).
  • the spacer was always 5'-AAAAC-3', except for the substrate of 5 nt that included the sequence 5'-AAAAA-3'.
  • the open reading frame of the HBV C gene was amplified from pCH9T/3091 (Nassal, M. J. Virol. 66, 4107-4116, 1992) using forward primer (5'-TATCTAAAGC TAGCTTCATG TCCTACTGTT)
  • the DNA product was cloned in the multiple cloning site of the plNDTM vector (Invitrogen) at the Nhe1 and Xho1 restriction sites.
  • the strategy for the design of the vector expressing the ribozymes included several steps : 1) Firstly, the vector pcDNA3TM (Invitrogen) was digested at the Hind III and Xho I sites removing a portion of the multiple cloning sites region; 2) Secondly, a cassette was synthesized using two overlapping oligodeoxynucleotides (5'-AGCTTGGTAC CGAGTCCGGA TATCAATAAA ATGC-3', SEQ ID NO: 19 and 5'-TCGAGCATTT TATTGATATC CGGACTCGGT ACCA-3', SEQ ID NO:20 allowing introduction of Knp I and EcoR V restriction sites followed by a poly(A) signal sequence.
  • Both ribozymes and RNA substrates were synthesized by run-off transcription from PCR products, Hind ⁇ linearized plasmid pHBV-1190 and Xba ⁇ linearized plasmid pHCVA templates. Run-off transcriptions were performed in the presence of purified T7 RNA polymerase (10 ⁇ g), RNAguardTM (32 units, Amersham Biosciences), pyrophosphatase (0.01 units, Roche Diagnostics) and linearized plasmid DNA in a buffer containing 80 mM HEPES- KOH, pH 7.5, 24 mM MgCI 2 , 2 mM spermidine, 40 mM DTT, 5 mM of each NTP and with or without 50 ⁇ Ci [ ⁇ - 32 P]UTP (New England Nuclear) in a final volume of 100 ⁇ l_ at 37 0 C for 3 hrs.
  • T7 RNA polymerase 10 ⁇ g
  • RNAguardTM 32 units, Amersham Biosciences
  • pyrophosphatase
  • reaction mixtures were treated with DNase RQ1 TM (Amersham Biosciences) at 37 ° C for 20 min, purified by phenoLchloroform extraction, and precipitated with ethanol.
  • DNase RQ1 TM Anagene-resistant DNA sequence
  • the viral RNA products and ribozymes were fractionated by denaturing 5% and 8%,
  • the reaction products were visualized by either UV shadowing or autoradiography.
  • the bands corresponding to the correct sizes of the ribozymes and the viral RNAs were cut out and eluted overnight at room temperature in a solution containing 0.5 M ammonium acetate and 0.1 % SDS.
  • the transcripts were desalted on Sephadex G-25TM (Amersham Biosciences) spun-columns, and were then precipitated, dissolved and quantified either by absorbance at 260 nm or 32 P scintillation counting.
  • RNA substrate (20 pmoles) was dephosphorylated using 0.2 units of calf intestinal alkaline phosphatase according to the manufacturer's recommendations (Roche Diagnostics). The reactions were purified by extracting with phenol:chloroform and precipitated with ethanol. Subsequently, the RNAs (10 pmoles) were 5 J -end labelled in a mixture containing 10 ⁇ Ci [ ⁇ - 32 P] ATP (3000 mCi/mmol; New England Nuclear) and 12 units of T4 polynucleotide kinase following the manufacturer's protocol (United States Biochemicals). The end-labelled RNAs were purified using denaturing PAGE, and the relevant bands excised from the gel, then eluted, precipitated, and dissolved in water.
  • Cleavage reactions for the mechanism analysis were carried out either with or without 5 ⁇ M of a facilitator (FCO, ⁇ '-AAAGTGAGAC AAGAA-3', SEQ ID NO:23), biosensor stem (BSO, 5'-TTCTTGTCTC ACTTT-3', SEQ ID NO:24) and an unrelated (UNO, 5'-CCCAATACCA CATCA-3', SEQ ID NO:25) oligodeoxynucleotide.
  • FCO ⁇ '-AAAGTGAGAC AAGAA-3', SEQ ID NO:23
  • biosensor stem BSO, 5'-TTCTTGTCTC ACTTT-3', SEQ ID NO:24
  • UNO 5'-CCCAATACCA CATCA-3', SEQ ID NO:25
  • Cleavage assays with the pools of mixed substrates were performed with trace amounts of radiolabeled substrates (50 000 cpm), non- labelled RNA substrate (2 ⁇ M) and SOFA-ribozymes (500 nM), except for the original ribozyme (WT, 2 ⁇ M). The reactions were incubated for 2 hours, analyzed on denaturing 10% Polyacrylamide gels, and revealed by PhosphorlmagerTM.
  • oligodeoxynucleotides (L3 1 : 5'-GCGAGGA-S'; P4': 5'-CCATCCG-S'; BS': 5'-TGTCTCA-3'; BL': 5'-TGAAACT- 3' and ST': 5'-CAGCTAG-3') 7 nt in size (10 pmol; 1 ⁇ L) were separately added to the samples before pre-incubating for another 10 min. Finally, 2 units of Esche ⁇ chia coli RNase H (Ambion; 1 ⁇ l_) were added to the mixtures and the samples incubated at 37°C for 10 min.
  • the reactions were quenched by adding 5 ⁇ L of cooled stop solution (97% formamide, 0.025% xylene cyanol and 0.025% bromophenol blue), the samples fractionated on denaturing 8% PAGE gels and the gels analyzed with a radioanalytic scanner (Storm TM).
  • cooled stop solution 97% formamide, 0.025% xylene cyanol and 0.025% bromophenol blue
  • HEK 293 EcR cells Human Embryonic Kidney
  • Dulbecco's modified Eagle's medium (DMEMTM ) (Sigma) supplemented with
  • RNA from HEK 293 EcR was extracted with Tri-Reagent (Bioshop Canada Inc, Burlington, Ontario, Canada). Northern blot analyses of total RNA (10 ⁇ g) extracted from HEK 293 EcR cells, were performed as described previously (D'Anjou, F., et al., J. Biol. Chem. 279, 14232-14239, 2004). The probes were synthesized as followed. For the HBV gene C probe, aliquot of the PCR product obtained previously (see DNA construct section) was cloned in the Xba I and Xho I sites of the pBlueScriptTM (SK) vector (Stratagene).
  • SK pBlueScriptTM
  • the resulting Sac I linearized vector was transcribed in vitro using T7 RNA polymerase in the presence of [ ⁇ -32P]UTP.
  • the ⁇ -actin RNA probe was synthesized using the Strip-EZTM RNA T7/T3 kit (Ambion) according to the manufacturer's conditions. All hybridizations were carried out for 16-18 h at 65°C. The membranes were exposed on PhosphorlmagerTM screen for 2-24 h. The densitometry analysis was carried out on ImageQuantTM software.
  • the SOFA-ribozyme of the present invention was tested using two accessible sites of the hepatitis B virus (HBV) RNA that have been previously selected for ribozyme cleavage ( ⁇ Rz-303 and ⁇ Rz-513)(Bergeron, LJ. , & Perreault, J. P. Nucleic Acids Res. 30, 4682-4691 , 2002). These ribozymes inefficiently cleaved an HBV-derived RNA of 1190 nucleotides (nt) (-15 %; Fig. 3).
  • HBV hepatitis B virus
  • the level of cleavage increases in proportion to the length of the biosensor, up to 10 nucleotides, at which point it decreased with increasing length (Fig. 6). Elongation of the biosensor stimulates the cleavage activity up to the point where product release becomes rate limiting. Astonishingly, in this specific experiment the SOFA + - ⁇ Rz-303-BS-10 performed four turnovers, while the original ribozyme only completed one. More importantly, the SOFA-ribozyme meets the classical criteria of an enzyme (e.g. it exhibits turnovers).
  • Fig. 6 the XC indicates the position of the xylene cyanol. The length of the bands in nucleotides is shown adjacent to the gel. The control (-) was performed in the absence of ribozyme.
  • oligodeoxyribonucleotide competition approach coupled with mutated ribozymes
  • Figs. 7A and 7B the roman numerals identify the steps of the mechanism.
  • Dashed lines identify oligodeoxyribonucleotide binding to either the substrate (i.e. FCO acting as facilitator) or the biosensor (BSO).
  • FCO oligodeoxyribonucleotide binding to either the substrate (i.e. FCO acting as facilitator) or the biosensor (BSO).
  • This oligodeoxyribonucleotide acts as a facilitator that renders the binding site more accessible to the catalytic region of the ribozyme.
  • the presence of the FCO does not alter the level of cleavage of SOFA + - ⁇ Rz-303 after an incubation of 3 hours, although it takes more time to reach this cleavage level.
  • One possible explanation is that the binding of both the biosensor and the P1 sequence favourably competes with the FCO for the substrate.
  • the specificity of a ribozyme can commonly be defined by the ability to discriminate between two or more similar RNA substrates.
  • two distinct experiments were performed. First, ten substrates were designed (see Table 3 below), each possessing an identical P1 binding sequence coupled to a distinct binding sequence for the biosensor. The substrates were successively extended at their 5' extremity by at least two nucleotides in order to provide them with assorted electrophoretic mobilities (Fig. 8A). Table 3 Substrates
  • RNA molecules that included an identical P1 binding sequence but different biosensor sequences.
  • a sequence of 7 nt long was retrieved twice in the HBV fragment (i.e. cleavage at positions 398 and 993; Figs. 8B and 8E), demonstrating the possibility of having multiple cleavage sites using the 7 nt requirement of a wild type ribozyme.
  • the classical HDV ribozyme did not allow the detection of cleavage at either of these sites, most likely because they were embedded in complex structures.
  • SOFA-ribozymes exhibited an efficient and specific cleavage at these sites (i.e. without any interference between the sites).
  • ribozymes D'Anjou, F., et al., J. Biol. Chem., 279, 14232-14239, 2004.
  • Figs. 8A and 8D there is reported cleavage assays of a pool of ten 5'- end-labelled substrates (a to j) by either specific SOFA-ribozymes (named A to J) or the original ribozyme (WT). All the ribozymes and substrates have a similar P1 stem sequence (P1), but differ in the biosensor sequences (BS). The length of each substrate is indicated in nucleotides.
  • FIGs. 8D indicate the positions of bromophenol blue and xylene cyanol, respectively.
  • the control (-) was performed in the absence of ribozyme.
  • Figs. 8B and 8E there are reported cleavage assays of the HBV-derived target by SOFA-ribozymes cleaving at either position 398 or 993.
  • Figs. 8C and 8F there are reported cleavage assays of a 1422-nt HCV-derived target by SOFA-ribozymes cleaving at either position 224 or 302 of the IRES.
  • the sequence of the P1 stem is identical at each site, but the biosensor sequences are different.
  • Fig. 9 The specific sequences of the various studied targeting sites derived from HBV and HCV viruses are illustrated in Fig. 9 (SEQ ID NO:36 to 41). The cleavage sites are indicated with arrows.
  • ribozyme H cleaves substrate d with a rate constant 15 000 times smaller than it does substrate h.
  • This first experiment confirms that a ribozyme cleaves its perfectly complementary substrate with a relatively high rate constant value, but that it is drastically less efficient for non-perfect couples (i.e. those including several mismatches).
  • a second experiment involving SOFA- ⁇ Rz-303 sequence variants with less potential for forming mismatches was performed. Twenty-three mutated ribozymes including 1 to 4 randomly distributed substitutions within the biosensor sequences were synthesized (Fig. 1 1 A, SEQ ID NO:58 to 81 ). A residue of the biosensor was substituted for by the same base found at the corresponding position within the substrate, thereby producing a mismatch.
  • cleavage activity of each mutated ribozyme was assessed, and the rate constant (k O bs) determined.
  • the k O bs are reported individually in panel A of Fig. 11
  • panel B illustrates the variation of the k Ob s average as a function of the number of mutations.
  • the decrease in the cleavage activity is directly related to the number of mutations (Fig. 11 B). While the presence of a single mismatch reduced the k Ob s values from 4 to 15 fold, the presence of 4 mutations yielded k Ob s values 18 to 106 fold smaller. The position of the mutation within the biosensor appeared to have only a small effect on the cleavage observed.
  • the SOFA- ribozyme adopts an inactive conformation, the off conformation. According to the SOFA design, this state is due to the 4 nucleotides blocker sequence binding the P1 region of the ribozyme, thereby preventing the binding of non ⁇ specific substrates (see Fig. 2). Consequently, the longer the blocker sequence, the better the "safety lock” effect.
  • SOFA-ribozymes with mutated blocker sequences were synthesized and their cleavage activities assessed by targeting the HBV-derived transcripts of 1190 nucleotides.
  • Fig. 12A A typical autoradiogram of a PAGE gel is illustrated in Fig. 12B.
  • SOFA- ⁇ Rz-303 was very active (i.e. BL-O, 81% cleavage). The same level of cleavage was detected in the presence of a 2 nucleotides blocker sequence (i.e.
  • Blockers of 6 nucleotides or more were also tested. In addition to blocking too much of the ribozyme in its inactive conformation (i.e. almost irreversible), we also observed ribozymes that self-cleaved the sequence adjacent to the blocker sequence (i.e. within the biosensor), an unacceptable phenomena.
  • the sequence of the blocker segment might also modulate the level of inhibition. We observed that if a mutated blocker cannot form a stem with the P1 strand, then no inhibition is observed. In contrast, previous experiments have shown that SOFA ' - ⁇ Rzs with different target sites on HBV-derived transcripts were all inactive (see previously). These ribozymes possessed the appropriate P1 strands and complementary blocker sequences, while their biosensor sequences could not bind the substrates. The inactivity of these SOFA ' - ⁇ Rzs confirmed that the blocker sequence plays its role by inhibiting the catalytic activity in the absence of the appropriate biosensor sequence. In all cases, the SOFA + - ⁇ Rzs possessing a biosensor sequence capable of binding the substrate efficiently cleaved their substrates.
  • a SOFA-ribozyme recognizes its substrate through two independent domains. Initially, the biosensor sequence binds its complementary sequence on the substrate, and, subsequently, the P1 stem is formed between the ribozyme and the substrate. In all experiments reported so far, the two binding domains were separated by 5 nucleotides simply to avoid the chance that the proximity and stacking of the P1 and biosensor would affect the release of the product. However, there was no scientific rational supporting this spacing of 5 nucleotides. In order to investigate this parameter seven model substrates possessing seven head-to-tail repetitions of the P1 stem domain (P1 N) followed by the SOFA- ⁇ Rz-303 biosensor sequence were synthesized (see Fig. 13A).
  • the substrates differed by possessing a distance of 0 to 6 nucleotides between the domain bound by the biosensor and the first adjacent P1 binding sequence. In this way we created the equivalent of 49 different substrates that included different spacer lengths.
  • the ribozyme should bind its complementary sequence at the 3' end of the substrate via its biosensor, and should subsequently find a P1 sequence at an ideal distance.
  • the cleavage experiments were performed during a short period of time (5 min) so as to permit only the unique cleavage reaction of 5' end labelled substrates to occur.
  • the substrates used in this experiment exhibited different electrophoretic mobilities depending on their sizes, which differed by one nucleotide.
  • the sequence of the stabilizer stem does not influence the ribozyme cleavage
  • the stabilizer brings both the 5' and 3' ends into a common terminal stem.
  • This domain has been included in the SOFA module due to previous observations revealing that the terminal P2 stem of the original ⁇ Rz provides tremendous stability to this RNA species (Levesque et al., RNA 8, 464-477, 2002). It was also shown above that the presence of the stabilizer within the SOFA-module increases the stability of SOFA- ⁇ Rz-303. Here, we address the influence of the stabilizer domain, which does not have an active role in the SOFA mechanism.
  • the SOFA-ribozyme that possesses this mutation has the same binding ability as the original, but does not display any cleavage activity.
  • Small oligodeoxynucleotides 7 nt in length complementary to various domains of the ribozyme were synthesized (Fig. 15A) and used with 5' end labelled SOFA- ribozyme in the absence (-) or presence (+) of its substrate.
  • the RNA-DNA heteroduplexes were monitored by ribonuclease H (RNase H) hydrolysis, an enzyme that cleaves the RNA strand of such heteroduplexes.
  • RNase H ribonuclease H
  • the oligodeoxynucleotide complementary to the L3 loop (L3') allowed the detection of a strong band of products in the absence of substrate, indicating that this region was single-stranded, in agreement with a previous report (Ananvoranich & Perreault, Biochem. Biophys. Res. Comm. 270, 600-607, 2000).
  • the addition of the substrate also led to the detection of this band at the same intensity, confirming that L3 is still single stranded. This observation is in contradiction to what has been observed in a previous study (Ananvoranich & Perreault, Biochem. Biophys. Res. Comm. 270, 600-607, 2000), but the experiments were performed here under different conditions than in the earlier report.
  • the oligodeoxynucleotide and the ribozyme were mixed together and incubated for 10 min prior to the addition of RNase H for the same period of incubation. These conditions favour the hybridization of the oligodeoxynucleotide to the L3 loop over the folding of the P1.1 stem that would release the oligodeoxynucleotide. Conversely, the oligodeoxynucleotide complementary to the P4 stem (P4') did not permitted the detection of any products of RNase H hydrolysis, confirming that this region is double-stranded.
  • the oligodeoxynucleotide complementary to the biosensor sequence permitted the detection of a relatively abundant RNase H product only in the absence of the substrate, indicating that this region was single-stranded within the off conformation. Only a trace amount of the hydrolysis product was detected upon the addition of the substrate, showing that in the on conformation the biosensor is bound to its substrate and thus is double-stranded.
  • oligodeoxynucleotide complementary to the blocker sequence gave the opposite pattern: no RNase H product was observed in the absence of the substrate, indicating that the blocker sequence was double- stranded (with the P1 strand of the ribozyme) within the off conformation; while cleavage product was detected in the presence of the substrate, showing that, under these conditions, the blocker was single-stranded. However, a small amount of product was detected, regardless of the length of the oligodeoxynucleotide tested (e.g. slightly longer).
  • a first experiment targeting an HBV derived transcript was performed in cultured cells (Fig. 16). Briefly, the HBV C gene open reading frame was subcloned in the inducible plNDTM vector (Invitrogen) (Fig. 16A). This vector contains five modified ecdysone response elements (E/GREs) and the minimal heat shock promoter for expression of RNA of interest. Using HEK-293 cells that stably express the ecdysone receptor by which the inductor ponasterone A enters the cell, the expression of the targeted RNA can be controlled.
  • E/GREs modified ecdysone response elements
  • RNA polymerase Il RNA polymerase Il
  • Fig. 16C illustrates an autoradiogram of the Northern blot hybridization demonstrating the success of the SOFA- ⁇ Rz activity to diminish the RNA target
  • RNA level In the presence of the original ⁇ Rz-303, only a weak reduction of the RNA level was observed. However, over 60% of RNA level reduction was observed in the presence of the SOFA + - ⁇ Rz-303 version. Conversely, in the presence of a
  • a SOFA-ribozyme lacking this domain (Fig. 18A, SEQ ID NO:88) was constructed.
  • the SOFA+- ⁇ Rz-NS (NS for no stabilizer) cleaved the HBV transcripts (SEQ ID NO:89) to the same extent as the original SOFA+- ⁇ Rz-303 while the corresponding SOFA ' -ribozyme (SOFA " - ⁇ Rz-NS) did not exhibit significant levels of cleavage activity (Fig. 18B).
  • the concept of a target-dependent module provides for a new generation of biosensorized ribozymes having a significantly improved substrate specificity and efficiency.
  • the on conformation implies that a ribozyme with a greater affinity for its substrate subsequently cleaves them faster. Meanwhile, the off conformation prevents cleavage of an inappropriate target, acting as a "safety lock".
  • the design of the specific on/off adapter was influenced by several factors. First, it is reminiscent of the human immune system, more specifically the cytotoxic T lymphocyte's activation mechanism. The T lymphocytes bind specific cell surface molecules which in turn dictate the T cell's responses.
  • the SOFA-ribozyme hybridizes to the RNA target (the activator) and specifically cleaves it.
  • the biosensor also remembers the mechanism of action of an oligodeoxynucleotide acting as facilitator for ribozyme cleavage.
  • the linkage of the biosensor directly to the ribozyme permitted a great gain, in terms of cleavage activity, compared to the use of two distinct molecules.
  • the blocker stem was influenced by the TRAP strategy (for Targeted Ribozyme-Attenuated Probe) in which a 3' terminal attenuator anneals to conserved bases in the catalytic core to form the off state of a hammerhead ribozyme.
  • the blocker domain of the SOFA module also inactivates the cleavage activity of the ribozyme by binding a sequence that is part of the catalytic core.
  • RNA species In fact, it has been shown that the ⁇ ribozyme was at least an order of magnitude more stable compared to a hammerhead ribozyme in cultured cells.
  • SOFA module is the fruit of a rational design. Using the Systematic Evolution of Ligands by Exponential enrichment (SELEX;
  • the TRAP ribozyme has demonstrated an activation of cleavage of as much as 1760 fold, with an average of more than 250 fold.
  • the SOFA-ribozyme higher than a 15 000 fold increase has been observed, with an average of more than 800 fold.
  • the SOFA system brings a two order of magnitude increase in the specificity to the ribozyme's action.
  • the SOFA concept appears to be a more efficient mode of increasing the substrate specificity of a ribozyme.
  • the biosensor In the presence of the desired substrate, the biosensor binds the complementary substrate sequence, leading in the release of the ribozyme's P1 stem from the blocker (Fig. 2).
  • the RNase H probing of the SOFA-ribozyme- substrate complex strongly suggest that the biosensor is base-paired with the substrate; while the blocker becomes susceptible to RNase H hydrolysis, indicating that it is single-stranded (Fig. 15).
  • Kinetic experiments have previously shown the optimal size of the biosensor to be 10 nucleotides. We demonstrated that each SOFA-ribozyme in our collection efficiently cleaved only the substrate containing the sequence complementary to its biosensor (see Fig. 10). Substrates that included sequence with several mutations in the binding region of the biosensor were poorly cleaved.
  • the SOFA-ribozymes harbour a stabilizer stem that joins the sequence found at the 5' and 3' ends into a stem (Fig. 2). This structure was confirmed by RNase H probing (Fig.15). In terms of mechanism, it appears clear that the stabilizer does not have an active role in the SOFA module (see Fig. 14) other than the improvement of the structure's stability.
  • the spacer sequence is not part of the SOFA-module, but it is an important parameter that influences the cleavage level.
  • the spacer is the sequence located between the substrate P1 strand domain and the sequence complementary to the biosensor
  • Fig. 2 It was shown that a minimal spacer of at least one nucleotide was preferable. Moreover, short spacer sequences (1 to 5 nucleotides) appeared to have higher levels of cleavage than did longer ones (see Fig. 13). Most likely the binding of the biosensor favours the subsequent formation of the P1 stem between the ribozyme and the substrate when the spacer is short.
  • the human genome is composed of 3 x 10 9 base pairs, of which -5% form mRNAs (i.e. 1.5 x10 8 bases). Therefore, the substrate specificity of a SOFA- ⁇
  • ribozyme is greater than 100 fold superior to what is needed to hit one site. This initiative provides confidence in the use of ribozymes in gene therapy and functional genomic applications, even if a mismatch is tolerated in the biosensor.
  • Fig. 19 illustrates one way to adapt the SOFA module to a cleaving hammerhead ribozyme, a cleaving hairpin ribozyme, a ligating hairpin ribozyme, or a cleaving DNazyme (i.e.
  • a proof-of concept has been performed with the DNazyme.
  • Cleavage assay were performed using a 5'-end 32 P-labelled substrate (S) of 46 nucleotides that generates a 5'-product of 23 nucleotides.
  • the DNazyme were purchased as DNA oligonucleotide and used directly in the experiments.
  • the reactions were performed and illustrated in Fig. 20, which shows an autoradiogram of a 6% denaturing PAGE gel of the cleavage assays.
  • the substrate was incubated alone (lane 1), with a DNazyme (lane 2), or with different versions of SOFA-DNazyme.
  • the SOFA module were assessed using separately either a good or irrelevant biosensor of 14 nucleotides in size (lanes 3 and 4, respectively), and a blocker sequence of 10 nucleotides (lane 5). Finally, SOFA module (i.e. including biosensor and blocker) were tested using both an appropriate biosensor (i.e. complementary to the substrate; SOFA+- DNazyme) and an irrelevant biosensor (i.e. not complementary to the substrate; SOFA " -DNazyme) (lanes 6 and 7, respectively). The original DNazyme cleaved a small radiolabeled substrate while a version harboring the blocker sequence was inactive (lanes 2 and 3). A SOFA + -DNazyme (i.e.
  • RNA silencing RNA
  • SiRNA silencing RNA
  • this technology can also be applied to other fields such as to siRNA or any other RNA implicated in a specific disease, its development or its spreading.
  • the SOFA can be made specific for such siRNA or other nucleic acid, acting as an on/off switch and improving substrates specificity, even if no enzymatic activity is involved such as with ribozymes.
  • the present invention can thus increase the popularity of siRNA which are these days often investigated as being a possible treatment for some conditions, but in life so far are not so often used due to their lack of specificity or to their immunogenicity.
  • the present invention can also be used with success in treatment for breast cancer to prevent transcription of the faulty genes, or in treatment of Alzheimer, preventing accumulation of irrelevant RNA.

Abstract

L'invention concerne un adaptateur d'acide nucléique dépendant de la cible, qui est lié à une séquence nucléotidique afin d'augmenter l'efficacité et la spécificité du clivage d'un acide ribonucléique en fonction de sa cible. Cet adaptateur comprend un biocapteur possédant un séquence spécifique complémentaire à une séquence cible d'un substrat, relié à une séquence tige de blocage complémentaire à une portion de la séquence nucléotidique. En l'absence de la séquence cible du substrat, la séquence tige de blocage forme une tige intramoléculaire avec l'acide nucléique relié, empêchant l'exposition de la séquence nucléotidique et verrouillant la séquence nucléotidique ainsi liée à l'adaptateur, dans une conformation inactive, et en présence de la séquence cible, la séquence tige de blocage se sépare de la séquence nucléotidique de manière à exposer la séquence nucléotidique liée à l'adaptateur, présentant ainsi une conformation active.
PCT/CA2005/001051 2004-07-06 2005-07-06 Adaptateur d'acide nucleique dependant de la cible WO2006002547A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP20050761763 EP1814896A4 (fr) 2004-07-06 2005-07-06 Adaptateur d'acide nucleique dependant de la cible
US11/631,689 US9012140B2 (en) 2004-07-06 2005-07-06 Target-dependent nucleic acid adapter
CA2632216A CA2632216C (fr) 2004-07-06 2005-07-06 Adaptateur d'acide nucleique dependant de la cible

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US58509304P 2004-07-06 2004-07-06
US60/585,093 2004-07-06

Publications (1)

Publication Number Publication Date
WO2006002547A1 true WO2006002547A1 (fr) 2006-01-12

Family

ID=35782454

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2005/001051 WO2006002547A1 (fr) 2004-07-06 2005-07-06 Adaptateur d'acide nucleique dependant de la cible

Country Status (4)

Country Link
US (1) US9012140B2 (fr)
EP (1) EP1814896A4 (fr)
CA (1) CA2632216C (fr)
WO (1) WO2006002547A1 (fr)

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007114986A2 (fr) * 2006-04-04 2007-10-11 Arcxis Biotechnologies, Inc. Sondes coopératives et leurs procédés d'utilisation
US7534588B2 (en) * 2007-05-25 2009-05-19 Asiagen Corporation Methods, kits and polynucleotides for simultaneously diagnosing viruses
WO2015035139A3 (fr) * 2013-09-06 2015-04-30 Prisident And Fellows Of Harvard College Nucléases cas9 commutables et leurs utilisations
US9322006B2 (en) 2011-07-22 2016-04-26 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9834791B2 (en) 2013-11-07 2017-12-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
US9938641B2 (en) 2006-12-18 2018-04-10 Fluidigm Corporation Selection of aptamers based on geometry
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100305197A1 (en) * 2009-02-05 2010-12-02 Massachusetts Institute Of Technology Conditionally Active Ribozymes And Uses Thereof
AU2013202354A1 (en) * 2012-06-18 2014-01-16 Speedx Pty Ltd Target detection and signal amplification

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5225337A (en) 1989-09-25 1993-07-06 Innovir Laboratories, Inc. Ribozyme compositions and methods for use
US5625047A (en) 1992-01-13 1997-04-29 Duke University Enzymatic RNA molecules
WO1999055856A2 (fr) 1998-04-29 1999-11-04 Universite De Sherbrooke Enzyme d'acide nucleique de coupure d'un arn

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050239061A1 (en) * 2000-03-01 2005-10-27 Marshall William S Identification and use of effectors and allosteric molecules for the alteration of gene expression
US20040009510A1 (en) * 2000-03-06 2004-01-15 Scott Seiwert Allosteric nucleic acid sensor molecules
WO2003052074A2 (fr) * 2001-12-14 2003-06-26 Yale University Reactifs de diagnostic d'acides nucleiques et procedes pour detecter des acides nucleiques, des polynucleotides et des oligonucleotides

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5225337A (en) 1989-09-25 1993-07-06 Innovir Laboratories, Inc. Ribozyme compositions and methods for use
US5625047A (en) 1992-01-13 1997-04-29 Duke University Enzymatic RNA molecules
WO1999055856A2 (fr) 1998-04-29 1999-11-04 Universite De Sherbrooke Enzyme d'acide nucleique de coupure d'un arn

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
ALAOUI-ISMAILI ET AL., ANTIVIRAL RES., vol. 46, 2000, pages 181 - 193
ANANVORANICH; PERREAULT, BIOCHEM. BIOPHYS. RES. COMM, vol. 270, 2000, pages 600 - 607
ANANVORANICH; PERREAULT, BIOCHEM. BIOPHYS. RES. COMM., vol. 270, 2000, pages 600 - 607
BERGERON ET AL., CURRENT MED. CHEM., vol. 10, 2003, pages 2589 - 2597
BERGERON L J ET AL: "Target-dependent on/off switch increases ribozyme fidelity.", NUCLEIC ACIDS RESEARCH, vol. 33, no. 4, February 2005 (2005-02-01), pages 1240 - 1248, XP003019025 *
BERGERON, L.J. ET AL., CURRENT MED. CHEM., vol. 10, 2003, pages 2589 - 2597
BERGERON, L.J.; PERREAULT, J.P., NUCLEIC ACIDS RES., vol. 30, 2002, pages 4682 - 4691
BURKE D H ET AL: "Allosteric hammerhead ribozyme TRAPs", BIOCHEMISTRY, vol. 41, no. 21, 28 May 2002 (2002-05-28), pages 6588 - 6594, XP003019026 *
D'ANJOU, F. ET AL., J. BIOL. CHEM., vol. 279, 2004, pages 14232 - 14239
LÉVESQUE ET AL., RNA, vol. 8, 2002, pages 464 - 477
LÉVESQUE, D. ET AL., RNA, vol. 8, 2002, pages 464 - 477
NASSAL, M. J., VIROL., vol. 66, 1992, pages 4107 - 4116
ROBERTSON M P ET AL: "In vitro selection of an allosteric ribozyme that transduces analytes to amplicons.", BIOTECHNOLOGY, vol. 17, no. 1, 1999, pages 62 - 66, XP002138120 *
ROY G ET AL: "Delta ribozyme has the ability to cleave in trans an mRNA.", NUCLEIC ACIDS RESEARCH, vol. 27, no. 4, 15 January 1999 (1999-01-15), pages 942 - 948, XP002119448 *
See also references of EP1814896A4 *
SILVERMAN S K ET AL: "Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA.", RNA, vol. 9, no. 4, April 2003 (2003-04-01), pages 377 - 383, XP002374942 *
SOUKUP G A ET AL: "Engineering precision RNA molecular switches.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, vol. 96, no. 7, 30 March 1999 (1999-03-30), pages 3584 - 3589, XP002158536 *
SOUKUP G A ET AL: "Nucleic acid molecular switches.", BIOTECHNOLOGY, vol. 17, no. 12, 1999, pages 469 - 476, XP002926497 *
WANG D Y ET AL: "A general approach for the use of oligonucleotide effectors to regulate the catalysis of RNA-cleaving ribozymes and DNAzymes.", NUCLEIC ACIDS RESEARCH, vol. 30, no. 8, 15 April 2002 (2002-04-15), pages 1735 - 1742, XP002429556 *
WILSON, DS; SZOSTAK, JW, ANNU. REV. BIOCHEM., vol. 68, 1999, pages 611 - 647

Cited By (60)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007114986A3 (fr) * 2006-04-04 2008-01-31 Arcxis Biotechnologies Inc Sondes coopératives et leurs procédés d'utilisation
AU2007233270B2 (en) * 2006-04-04 2013-06-20 Fluidigm Corporation Cooperative probes and methods of using them
WO2007114986A2 (fr) * 2006-04-04 2007-10-11 Arcxis Biotechnologies, Inc. Sondes coopératives et leurs procédés d'utilisation
US9938641B2 (en) 2006-12-18 2018-04-10 Fluidigm Corporation Selection of aptamers based on geometry
US7534588B2 (en) * 2007-05-25 2009-05-19 Asiagen Corporation Methods, kits and polynucleotides for simultaneously diagnosing viruses
US9322006B2 (en) 2011-07-22 2016-04-26 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9359599B2 (en) 2013-08-22 2016-06-07 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10227581B2 (en) 2013-08-22 2019-03-12 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
EP3693461A1 (fr) * 2013-09-06 2020-08-12 President And Fellows Of Harvard College Nucléases cas9 commutables et leurs utilisations
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
WO2015035139A3 (fr) * 2013-09-06 2015-04-30 Prisident And Fellows Of Harvard College Nucléases cas9 commutables et leurs utilisations
US9340799B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College MRNA-sensing switchable gRNAs
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US9737604B2 (en) 2013-09-06 2017-08-22 President And Fellows Of Harvard College Use of cationic lipids to deliver CAS9
US9526784B2 (en) 2013-09-06 2016-12-27 President And Fellows Of Harvard College Delivery system for functional nucleases
US9228207B2 (en) 2013-09-06 2016-01-05 President And Fellows Of Harvard College Switchable gRNAs comprising aptamers
US9388430B2 (en) 2013-09-06 2016-07-12 President And Fellows Of Harvard College Cas9-recombinase fusion proteins and uses thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US9340800B2 (en) 2013-09-06 2016-05-17 President And Fellows Of Harvard College Extended DNA-sensing GRNAS
US11390887B2 (en) 2013-11-07 2022-07-19 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US10640788B2 (en) 2013-11-07 2020-05-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAs
US10190137B2 (en) 2013-11-07 2019-01-29 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US9834791B2 (en) 2013-11-07 2017-12-05 Editas Medicine, Inc. CRISPR-related methods and compositions with governing gRNAS
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US9840699B2 (en) 2013-12-12 2017-12-12 President And Fellows Of Harvard College Methods for nucleic acid editing
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Also Published As

Publication number Publication date
CA2632216A1 (fr) 2006-01-12
EP1814896A4 (fr) 2008-07-30
US20080268516A1 (en) 2008-10-30
US9012140B2 (en) 2015-04-21
CA2632216C (fr) 2013-11-12
EP1814896A1 (fr) 2007-08-08

Similar Documents

Publication Publication Date Title
US9012140B2 (en) Target-dependent nucleic acid adapter
JP2021118714A (ja) Ｃｒｉｓｐｒ系組成物及び使用方法
Sun et al. Catalytic nucleic acids: from lab to applications
Baum et al. Deoxyribozymes: useful DNA catalysts in vitro and in vivo
JP6752957B2 (ja) オリゴヌクレオチドの産生のための新規プロセス
JP5795563B2 (ja) アプタマー調節される核酸及びその利用
AU717736B2 (en) Catalytic nucleic acid and its medical use
CN110709514B (zh) 使用具有串联重复序列的供体dna修复模板的位点特异性dna修饰
US20060223777A1 (en) Highly functional short hairpin RNA
EP1105513A2 (fr) Procedes et compositions utilisables dans un trans-epissage d'arn induit par une "epissageosome"
Taylor et al. A modular XNAzyme cleaves long, structured RNAs under physiological conditions and enables allele-specific gene silencing
JP2002514080A (ja) 酵素ｄｎａ分子
US20060134658A1 (en) Use of RNA trans-splicing for generation of interfering RNA molecules
Liu et al. Suppression of platelet-type 12-lipoxygenase activity in human erythroleukemia cells by an RNA-cleaving DNAzyme
Ramesh et al. Metabolite-binding ribozymes
Hélène et al. Sequence-specific artificial endonucleases
Bergeron et al. Functional characterization of the SOFA delta ribozyme
US6307041B1 (en) Circular, hairpin, circular/hairpin, lariat, and hairpin-lariat hammerhead ribozymes
Pan et al. Catalytic DNAzymes: derivations and functions
Lützelberger et al. Strategies to identify potential therapeutic target sites in RNA
Chen A novel single-stranded DNA expression vector
Takagi-Sato et al. Fine-Tuning of ENA® Gapmers as Antisense Oligonucleotides for Sequence-Specific Inhibition
Kundu Directed Evolution of a Novel Heterochiral Ribonuclease Ribozyme and Kinetic Characterization of Heterochiral DNA Strand Displacement Reactions
Noé Mata Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells
Kim et al. Efficient target site selection for an RNA-cleaving DNAzyme through combinatorial library screening

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

WWE Wipo information: entry into national phase

Ref document number: 2005761763

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2005761763

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2632216

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 11631689

Country of ref document: US